An aldehyde-crosslinking mitochondrial probe for STED imaging in fixed cells

Significance Super-resolution stimulated emission depletion (STED) imaging of mitochondrial ultrastructure was mostly demonstrated with live cells due to the limited availability of fixable probes. The development of a mitochondrial probe that is both optically outstanding and retaining strong fluorescence signals after fixation would further democratize super-resolution imaging techniques for mitochondrial research. To address this challenge, we employ an emerging bioconjugation strategy to develop an aldehyde-fixable mitochondrial probe, PK Mito Orange FX (PKMO FX), which enables nanoscopic STED imaging both before and after fixation. With PKMO FX, we demonstrate multiplexed imaging of mitochondrial cristae and various cellular organelles, as well as correlative super-resolution light and electron microscopy.


Materials and methods
Absorption and fluorescence spectroscopy PKMO FX for spectroscopy were prepared as stock solutions (1 mM) in DMSO and diluted with acetonitrile (ACN) to 1 μM.Spectroscopy was performed using a 1cm square quartz cuvette.All measurements were taken at room temperature (25 ± 2 °C).Absorption and fluorescence emission spectra of the sample solution were measured using a Duetta spectrometer (HORIBA Instruments, Japan).Normalized spectra are shown for clarity.

PKMO FX labeling and fixation
For optimal results, we recommend optimizing PKMO FX concentration and staining duration for each cell line.In general, for HeLa or COS-7 cells, we recommend staining the cells with 100-700 nM PKMO FX for 15 min -2 h.Verapamil is not essential for the staining, but 6 -10 µM verapamil can be added to reduce heterogeneity of the staining.The cells were washed with fresh PBS buffer (pH =7.4) twice after staining and fresh medium was added into the dish for live-cell imaging.If the signal is noisy with dissatisfactory mitochondrial contrast, we recommend longer wash time in DMEM.After live-cell imaging of PKMO FX, the cells were washed with fresh preheated (37 °C) PBS buffer (pH =7.4) three times and fixed with preheated (37 °C) 2% -2.5% GA or 4% FA for at least 10 min.Specific staining conditions in this work: QUAD scanning microscope (Abberior Instruments).The microscope was equipped with a UPlanSApo 100×/1.40Oil [infinity]/0.17/FN26.5objective (Olympus).PKMO FX was excited by a 561 nm laser and depleted with a 775 nm STED laser.Deconvolution was done using Abberior Imspector software.

Measure of fluorescence retention ratio of different mitochondrial dyes after fixation
To simulate the real situation in actual use, HeLa cells were stained with the optimal or recommended staining conditions for each dye: 1. Cy3.5-NH2 (PKMO FX): 600 nM plus 8 μM verapamil for 80 min.2. Cy5-NH2: 500 nM plus 8 μM verapamil for 60 min.3. Mito Tracker Red: 200 nM for 20 min.4. Mito Tracker Deep Red: 200 nM for 20 min.5. Cy3.5-NHS: 600 nM plus 8 μM verapamil for 60 min.6. Cy3.5-CHO: 1 μM plus 8 μM verapamil for 60 min.After staining, live cell imaging was performed in one selected area (80 × 90 μm, contains about 4 -7 cells) under a 100 × confocal microscope.Then the cells were fixed with GA (final concentration was 2%) without moving the dish.The fixed cells of the same area were imaged 10 min after fixation.The laser intensity was consistent with that of live cell imaging.For one dye, the experiment was repeated three times.For each image, mitochondria were first sorted out by ImageJ: Image-Adjust-Threshold function, then the fluorescence intensity of mitochondria was counted.The fluorescence retention ratio is calculated as: fixed-cell mitochondrial intensity / living-cell mitochondrial intensity.

EdU-based metabolic labeling
For the metabolic labeling, the Click-iT Plus EdU Imaging Kit (C10337, Thermo Fisher Scientific) was used according to the manufacturer protocol but without using a permeabilization agent.HeLa cells were incubated with both PKMO FX (600 nM plus 10 μM verapamil) and EdU (50 µM) for 2 hours.After that the cells were washed with PBS buffer three times and fixed with 2% GA.The fixative was removed and the sample was washed twice with blocking solution (3% BSA in PBS).For EdU detection, the Click-iT Plus reaction cocktail including reaction buffer, copper protectant, Alexa Fluor 488 picolyl azide, and reaction buffer additive was freshly prepared and the sample was incubated for 30 minutes with this solution.Sample was washed several times with 3% BSA in PBS before imaging.Immunolabeling Immunolabeling for TOM20: HeLa cells were stained with DMEM supplemented with 650 nM PKFO and 10 µM Verapamil for 1 h at 37°C and 5% CO2.Before fixation, cells were washed with pre-warmed (37 °C) DMEM three times and then prefixed by immersion with pre-warmed 2% GA in PBS buffer (pH 7.4) for 15 min.The fixative was washed by PBS buffer three times, then treated the cells with 0.5% Triton X-100 and 5% BSA in PBS buffer for 1 h.All following steps were performed in PBS.Cells were then incubated with rabbit-derived primary antibodies anti-TOM20 (ABclonal, Cat.No. A19403, 1:75) for 1 h at RT, followed by washed with PBS buffer for 5 min x 3 times.Then the cells were incubated with secondary antibodies (goat anti-rabbit) with abberior STAR RED fluorophore (Abberior, Göttingen, Germany, STRED-1002, 1:75) for 1h.Samples were washed 5 min x 3 times, post-fixed with 2% GA, mounted in PBS buffer and recorded under STED microscope the same day.Immunolabeling for Actin, ATPB, and Tubulin: COS-7 cells were stained with DMEM supplemented with 600 nM PKFO and 10 µM Verapamil for 2 h at 37°C and 5% CO2.Before fixation, cells were washed with pre-warmed (37 °C) DMEM three times and then prefixed by immersion with pre-warmed 2% glutaraldehyde (Agar Scientific, Stansted, UK, Cat.No. AGR1020) in 0.1 M phosphate buffer (pH 7.4) for 20 sec.The fixative was then replaced with pre-warmed 4% formaldehyde (thermo scientific, Rockford, IL, USA, Cat.No. 28908) in 0.1 M phosphate buffer (pH 7.4) and fixation was continued for 8 min at RT.The fixative was replaced by 0.1 M phosphate buffer and the cells were kept at RT for 10 min, followed by incubation with 0.1 M NH4Cl in 0.1 M phosphate buffer for 5 min.Cells were permeabilized with 0.25% Triton X-100 in 0.1 M phosphate buffer for 5 min and washed 5 times with phosphate buffered saline (PBS, 137 mM NaCl, 2.68 mM KCl, and 10 mM Na2HPO4, pH 7.4).All following steps were performed in PBS.For labeling of F-actin, cells were incubated with Phalloidin-Abberior STAR 635 (Abberior GmbH, Göttingen, Germany, Cat.No. ST635-0100-20UG, 1:50) for 2h at RT. Labeling of microtubules and mitochondria was achieved by incubation with primary antibodies against alpha-tubulin (Merck, Cat.No. T6074-100UL, 1:700) and ATPB (Abcam, Cambridge, UK, Cat.No. ab5432, 1:100) for 1h at RT. Cells were washed five times and primary antibodies were detected by incubation with secondary sheep-anti-mouse antibodies (Jackson Immuno Research Laboratories, West Grove, PA, USA) custom-labeled with Abberior STAR RED NHS-ester (Abberior GmbH) for 1h at RT.Samples were washed 5 times, mounted in PBS or mounting medium (Ibidi GmBH, Gräfelfing, Germany, Cat.No. 50001) and recorded the same day.Pre-embedding CLEM Cells were grown in glass bottom dishes (MatTek, # P356-1.5-14-C)which were coated with a carbon finder pattern using a mask (Leica, # 16770162) and a carbon coater ACE 200 (Leica).Cells were stained with 650 nM PKMO FX plus 8 μM verapamil for 1.5 h.Cells were fixed for 15 min at room temperature in 2% glutaraldehyde (Sigma, # G5882-100ML) with 2.5 % sucrose (Roth, # 4621.1) and 3mM CaCl2 (Sigma, # C7902-500G) in 0.1M HEPES buffer pH 7.4 (Roth, # 9105.1).Cells were washed three times with 0.1M HEPES buffer and fluorescent and brightfield images were taken using the SP8 gSTED microscope (Leica) with 100x/1.40 oil objective.Localization coordinates of cells of interest were noted.Cells were incubated with 1% Osmium tetroxide (Science Services, # E19190) and 1.5% potassium hexacyanoferrate (Sigma, # P8131) for 30 min at 4°C.After 3 × 5min wash with 0.1M Cacodylate buffer (Applichem, # A21400100), samples were dehydrated using ascending ethanol series (50%, 70%, 90%, 100%) (VWR, # 153386F) for 7 min each at 4°C.Cells were infiltrated with a mixture of 50% Epon/ethanol for 1 h, 66% Epon/ethanol for 2 h and with pure Epon (Sigma, # 45359-1EA-F) overnight at 4°C.TAAB capsules filled with Epon were placed upside down onto the glass bottom and cured for 48 h at 60°C.Glass bottom was removed by alternatingly putting the dish into boiling water and liquid nitrogen.Block face was trimmed to the previous noted square using a razor blade and ultrathin sections of 70 nm or 300 nm for tomography were cut using an ultramicrotome (Leica Microsystems, UC6) and a diamond knife (Science Services # DU3530) and stained with 1.5 % uranyl acetate (Agar Scientific, # R1260A) for 15 min at 37°C and 3% Reynolds lead citrate solution made from Lead (II) nitrate (Roth, # HN32.1) and tri-Sodium citrate dehydrate (Roth #4088.3)for 3 min.
Images were acquired using a JEM-2100 Plus Transmission Electron Microscope (JEOL) operating at 80kV or at 200kV for tomography equipped with a OneView 4K camera (Gatan).Tomograms of 300nm thick sections were generated using SerialEM(1)and IMOD (2).Overlay of TEM and STED images were generated using the EC-CLEM plugin (3) for the software ICY.

Fig S8. Comparison of mitochondrial morphology of HeLa cells stained with PKMO FX at different concentrations.
HeLa cells were stained with x nM PKMO FX plus 10 μM verapamil for 2 h.From a to f, x =162.5, 325, 650, 1300, 2600, and 5200 nM respectively.Concentrations exceeding 1300 nM were found to induce noticeable mitochondrial swelling and roundness.Therefore, we recommend depending on the used cell line a staining concentration between 150-700 nM, an incubation time between 15 min and 2 h and optionally the addition of verapamil.

Fig
Fig S9.STED image of a HeLa cell stained with PKMO FX, 13 days after staining and fixation.PKMO FX offers long-term and stable cross-linking which enables STED imaging over 10 days after fixation.HeLa cells were labeled with PKMO FX and then fixed with 2% GA for 15 min.Cells are washed with ddH2O and stored under 4℃ before repeatedly imaged.The mitochondrial cristae are reproducibly visible with STED nanoscopy after 13 days.Scale bar = 2 μm.

Fig
Fig S10.Live-cell STED imaging under different depletion laser powers of HeLa cells stained with PKMO FX or MTR.a. Raw STED images and analysis of FWHM of fluorescence intensity line profiles recorded from HeLa cells stained with PKMO FX.HeLa cells were stained with 600 nM PKMO FX for 60 min, then imaged using different depletion laser powers from 50% to 90%, respectively.Fluorescence intensity line profiles measured across mitochondrial cristae (from the yellow line area in the white dotted box) are shown on the right of each image.Zoomed-in images of the mitochondria from the yellow line area are shown on the upper left of each image.The FWHM was calculated from a Gaussian fitting within the STEDYCON software, Abberior Instruments.Scale bars = 2 μm.b.Raw STED images and analysis of FWHM of fluorescence intensity line profiles recorded from HeLa cells stained with MTR.HeLa cells were stained with 250 nM MTR for 30 min, then imaged using different depletion laser powers from 50% to 90%, respectively.Fluorescence

Fig
Fig S11.MTDR is not suitable for both live-cell and post-fixation STED imaging.a. Raw STED images of HeLa cells stained with MTDR in the absence of verapamil.HeLa cells were stained with 200 nM MTDR for 20 min and fixed with 2.5% GA.Images were taken before fixation and 10 min after fixation.Scale bars = 2 μm.b.Raw STED images of HeLa cells stained with MTDR in the presence of verapamil.HeLa cells were stained with 500 nM MTDR plus 8 μM verapamil for 60 min and fixed with 2.5% GA.Images were taken before fixation and 10 min after fixation.Scale bars = 2 μm.

Figure S12 .
Figure S12.Comparison of FA/GA fixatives on mitochondrial morphology.a. FA fixation can cause significant swelling and roundedness (highlighted with white arrow) in

Fig S14 .
Fig S14.Comparison of SBR and STED imaging performance of PKMO FX, MTR and MTDR after fixation by 4% FA plus 0.1% GA. a. Confocal images of HeLa cells stained with PKMO FX.Images of same area were taken before and after fixation.Scale bars = 10 μm. b.STED images of HeLa cells stained with PKMO FX.Images of same area were taken before and after fixation.Scale bars = 2 μm.c.Confocal images of HeLa cells stained with MTR.Images of same area were taken before and after fixation.Scale bars = 10 μm.d.STED images of HeLa cells stained with MTR.Images of same area were taken before and after fixation.Scale bars = 2 μm.e. Confocal images of HeLa cells stained with MTDR.Images of same area were taken before and after fixation.Scale bars = 10 μm.f.STED images of HeLa cells stained with MTDR.Images of same area were taken before and after fixation.Scale bars = 2 μm.